Characterisation of ER-beta functions in prostate cancer (ChIP-Seq)

To investigate the effect of a novel ER-beta selective ligand alone and with Enzalutamide on AR, ARv7, MYC genomic binding and H3K27ac distribution in prostate cancer cell lines Overall design: Cells were treated cells in the presence of OSU-ERb-12 alone (100 nM) or E2 alone (100nM), Enza (1 microMolar) or the combinations for 6h. Briefly, approximately cells were adsorbed to concanavalin A beads (Epicypher), and washed (150mM NaCl, 20mM HEPES, 0.01% Digitonin, 0.5mM Spermidine, protease inhibitor cocktail EDTA-free mini tab 1tab/10mL (Roche)) to permeabilize cells and nuclei. Samples were incubated overnight with antibodies against IgG (2729, Cell Signaling Technology), H3K27 (8173, Cell Signaling Technology), AR (5153, Cell Signaling Technology), ARv7 (ab198394, abcam), MYC (9402, Cell Signaling Technology) for 16 hours, and antibody bound DNA was then enzymatically cut with pAG-MNase (Epicypher). Samples were treated with Stop Buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 50ug/mL RNase A, 50ug/mL Glycogen) to prevent further enzymatic activity. DNA fragments released into supernatant were collected and purified by Monarch PCR and DNA clean-up kit (New England Biosciences). Comparative AR, ARv7, MYC and H3K27ac cistrome analsyes by ChIP-seq analsyes of prostate cell lines treated with OSU-ERb-12 alone (100 nM) or E2 alone (100nM), Enza (1 microMolar) or the combinations for 6h